The disclosure relates generally to the field of immunology and allergic reactions.
Poison ivy is one of the most medically problematic plants, and is responsible for causing allergic contact dermatitis in humans. It has been estimated that approximately 85% of the US population is sensitized to these plants. While the chemistry of the allergen and the immunologic mechanism of the reaction are different, both clinical experience and animal studies suggest that poison ivy, like respiratory allergy to ragweed, should be responsive to injection immunotherapy. Ragweed allergen was identified and quantitated in the 1960's and its safe and effective immunotherapy doses identified in dose ranging studies in the 1960‘s and’70's. Immunotherapy has the prospective ability to lessen the abnormal immunological responses to an allergen. Double blind immunotherapy discontinuation studies of ragweed have shown that even after a full year patients show no symptoms of response to the plant. There are no known studies to validate human injection immunotherapy or identify safe and effective treatment doses and schedules for poison ivy allergies.
Urushiol is an oily organic allergen found in plants in the family Anacardiaceae. Notable plants includes cashew (in the type genus Anacardium), mango, pistachio, poison ivy (e.g., Toxicodendron radicans), poison oak (e.g., Toxicodendron diversilobum and Toxicodendron pubescens), sumac, smoke tree, marula, yellow mombin, and cuachalalate. It has been demonstrated that the heteroolefinic components of the urushiols contained in the plants within this family are not identical in structure or composition. The term urushiol as used herein includes mixtures of heteroolefinic catechols of Anacardiaceae plants, e. g., poison ivy, poison oak, cashew, pistachio, or mango, as well as to individual components of such mixtures.
Urushiol is a mixture 3-n-alk-(en)-yl catechols with varying degrees of unsaturation on either the C15 side chain (derived from poison ivy chain) or C17 side chain (derived from poison oak), often including those shown in FIG. 1. Conclusive identification of the structure was first made in 1934; purification yielded a yellow oil with a boiling point of 210 degrees Celsius. Urushiol primarily resides in resin canals, present in the leaves, flowers, roots, bark and fruits of the plant. In addition to poison oak, poison ivy, and poison sumac, other urushiol-containing plants in this family are the Chinese and Japanese lac trees (R. verniciflua L.) and the Yunnan, Formosan and Indo-Chinese lac trees (R. succedanea L.). Agriculturally significant Mango (M. indica), Cashew (A. occidentale), and Pistachio (P. vera) trees also contain urushiol.
The long aliphatic side chains of these catechols cause the molecules to be hydrophobic, making urushiol one of the most lipophilic haptens that cause contact dermatitis. As a result of this lipophilicity urushiol tends to concentrate in cellular membranes. Upon contact with human tissue, the catechol ring undergoes oxidation, forming an electrophilic o-quinone. This undergoes a regiospecific attack by model sulfhydryl and amino nucleophiles present on proteins to form the adduct. However, the lack of covalent bonding between urushiol and cell membranes used for introduction into cultures makes the mechanism of antigen presentation unclear.
In vivo experiments suggest that T-cell receptors may be directed against the side chain, with the di-saturated and tri-saturated chains producing the majority of the immunological response. Yet due to the hydrophobic nature of the molecule, the alkyl side chain should be buried within the membrane leaving the catechol ring near the surface. Therefore it is difficult to theorize how the side chain could move out of the membrane and have an effect upon immunological response. One proposed mechanism attributes it to a membrane vesicle. Alternatively T-cell reaction could be against the catechol ring, and increasing reactivity with unsaturated side chains could be due to an altered presentation of the catechol ring.
Immunotherapy with water-soluble ragweed antigen E has been shown to not only prevent reactions on natural exposure during immunotherapy but also to provide durable protection for at least a year after stopping treatment. Double blind immunotherapy discontinuation studies of ragweed have shown that, even after a full year, patients show no symptoms of response to the antigen. Based on immunological studies of ragweed, it was hypothesized that urushiol can be extracted and used in immunotherapy against contact dermatitis. However, previous extraction methods have proven difficult and inefficient, being multi-stepped and involving multiple solvents. The most common technique used by others involves soaking plant material in ethanol followed by distillation or dry packed silica column chromatography. Urushiol congeners can be separated using HPLC if desired. An example of an urushiol extraction method (hereinafter the “ElSohly method”) described by ElSohly et al. (J. Nat. Prod., 1982, 45(5):532-538) in summarized in FIG. 2.
The ElSohly method and other multiple-solvent purification methods have shortfalls, including use of undesirably large quantities of solvents, restriction to small (volumetric) scale processing, and low yields. Limitations of crude ethanol extracts as allergy vaccines include limited storage stability and urushiol concentrations insufficient for effective treatment of moderately sensitive individuals.
Crude ethanol extracts having urushiol concentrations up to 1-2 milligram per milliliter have been described. (Coifman R E, “Successful immunotherapy for poison ivy,” presented to 2010 Annual Meeting of the American Academy of Allergy Asthma & Immunology, New Orleans La., 28 Feb. 2010 (Abs. 142), epub J. Allerg. Clin. Immunol. 2010 (February)).
A need exists for improved methods for extraction of urushiol from plant materials at concentrations and purity levels suitable for immunotherapy or other treatment of hypersensitivity.